1. Field of the Invention
This invention relates to the gyrase B gene which encodes for the subunit B protein of DNA gyrase, a type II topoisomerase that catalyzes the negative supercoiling of bacterial DNA, sequence polymorphisms in the Campylobacter gyrB gene, and species-specific PCR (polymerase chain reaction) assays and PCR-RFLP (PCR-restriction fragment length polymorphism) using the restriction enzymes DdeI, XspI, or the combination of MboI and HindIII for differentiation of Campylobacter species, and a method of speciating Campylobacter. 
2. Description of the Relevant Art
Campylobacter spp. are the most common cause of bacterial gastrointestinal infection in the United States, Japan, and other developed nations. Infections have the highest incidence in infants, young children, and in adults 20 to 40 years of age. Travel to developing countries is a major risk factor for acquiring Campylobacter infections. The majority of human infections due to Campylobacter spp. are sporadic or occur in small family clusters rather than large outbreaks, rendering identification of sources of infection through epidemiological investigations difficult. There are numerous animal reservoirs for Campylobacter spp., including cattle, sheep, poultry, and swine; however, the major animal source for sporadic infections is poultry (Corry et al. 2001. J. Appl. Microbiol. 90:96S-114S; Manning et al. 2003. Appl. Environ. Microbiol. 69: 6370-6379; Nielsen, E. M. 2002. Lett. Appl. Microbiol. 35: 85-89). A recent population-based, case-control study conducted by Friedman et al. (2004. Clin. Infect. Dis. 38 (Suppl 3): 285-296) indicated that consuming poultry, particularly prepared in restaurants, is a major risk factor for sporadic human Campylobacter infection in the U.S. Household pets, including dogs and cats, are also a source of Campylobacter infections (Damborg et al. 2004. J. Clin. Microbiol. 42: 1363-1364; Moser et al. 2001. J. Clin. Microbiol. 39: 2548-2557). In addition to animal sources, contaminated vegetables and shellfish have also been linked with Campylobacter infection (Altekruse et al. 1994. J. Am. Vet. Assoc. 204: 57-61; Jacobs-Reitsma, W. 2000. In: Campylobacter, Nachamkin and Blaser, eds., ASM Press, Washington, D.C., pages 467-481), and contaminated water supplies have been implicated in point-source outbreaks (Goossens et al. 1995. J. Infect. Dis. 172: 1298-1305; Hanninen et al. 2003. Appl. Environ. Microbiol. 69: 1391-1396).
The genus Campylobacter consists of 16 species and six subspecies (On, S. L. W. 2001. J. Appl. Microbiol. 90: 1S-15S). Some species mainly cause disease in animals, including cattle, swine, sheep, dogs, and cats (Lastovica et al. 2000. In: Campylobacter, Nachamkin and Blaser, eds., ASM Press, Washington, D.C., pages 89-120). The thermophilic species, C. jejuni, C. coli, C. lari, and C. upsaliensis, but in particular C. jejuni, account for the majority of human infections; however, other species have been linked with diarrheal illness, periodontal disease (C. concisus, C. gracilis, C. rectus, and C. showae), meningitis, and septicemia in humans (Lastovica et al., supra). As examples, C. lari was associated with a water-borne outbreak of gastroenteritis (Borczyk et al. 1987. Lancet 1: 164-165), C. upsaliensis caused an outbreak in four day care centers in Brussels, affecting 44 children (Goossens et al., supra), C. jejuni and C. fetus subsp. fetus caused an outbreak associated with raw milk in individuals who attended a banquet in Wisconsin (Klein et al. 1986. JAMA 255: 361-364), and a number of different species have been isolated from stools of diarrheic patients (Lastovica et al., supra). Because of technical limitations in current cultural and phenotypic methods employed for detection, isolation, and typing of Campylobacter, non-jejuni species are likely under-reported in clinical specimens. Further research is needed to identify sources of infection, routes of transmission, and disease syndromes associated with non-jejuni Campylobacter species.
A number of methods have been described for detection and speciation of Campylobacter, including 16S rRNA sequence analysis (Gorkiewicz et al. 2003. J. Clin. Microbiol. 41: 2537-2546) and PCR-based assays for detection of single species or for species differentiation based on rRNA genes (Junior et al. 2003. Pesqui. Odontol. Bras. 17: 142-146, 21). Real-time PCR assays using fluorescence resonance energy transfer (FRET) probes targeting 16S rRNA sequences in Campylobacter spp. followed by melting peak analysis were used for detection and identification of different species (Logan et al. 2001. J. Clin. Microbiol. 39: 2227-2232). A reverse hybridization line probe assay based on use of species-specific probes targeting a putative GTPase could distinguish C. jejuni, C. coli, C. lari, and C. upsaliensis (van Doorn et al. 1999. J. Clin. Microbiol. 37: 1790-1796). On and Harrington (2000. FEMS Microbiol. Lett. 193: 161-169) distinguished Campylobacter species using an amplified fragment length polymorphism (AFLP)-based technique. However, the complex nature of the AFLP patterns that were generated rendered interpretation of results difficult, and the high cost of the equipment required may preclude the use of this technique in many research laboratories. Recently, Mandrell et al. (2005. Appl. Environ. Microbiol. 71: 6292-6307) described a method for speciating C. coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis using matrix-assisted laser desorption ionization-time of flight mass spectrometry. A PCR-microarray method based on PCR amplification of Campylobacter species-specific genes and rRNA regions followed by hybridization to immobilized probes has been developed (Kerama et al. 2003. Mol. Cell Probes 17: 187-196; Volokhov et al. 2003. J. Clin. Microbiol. 41: 4071-4080).
Restriction enzyme analysis of PCR amplicons, known as PCR-restriction fragment length polymorphism (PCR-RFLP), is a useful tool for molecular characterization of food-borne pathogens, including differentiation of thermophilic campylobacters (Engvall et al. 2002. J. Appl. Microbiol. 92: 47-54). After amplification, the PCR product is digested using one or more restriction enzymes to produce fragments of specific sizes based on the DNA sequence of the gene. The PCR-RFLP technique based on the flagellar flaA and/or flaB genes has been used for speciation and subtyping of Campylobacter strains (Harrington et al. 2003. J. Appl. Microbiol. 95: 1321-1333; Koenraad et al. 1995. Epidemiol. Infect. 115: 485-494; Stern et al. 1997. Avian Dis. 41: 899-905). Intra- and inter-genomic recombination of the flaA and flaB genes, however, may contribute to the variability seen when this method is used (Harrington et al. 1997. J. Clin. Microbiol. 35: 2386-2392). The development of genotypic methods with the ability to precisely discriminate among the different species of Campylobacter is essential for effective monitoring and surveillance to determine the prevalence of these organisms in the environment and for defining the epidemiology of human infections.
The gyrase B gene encodes for the subunit B protein of DNA gyrase, a type II topoisomerase that catalyzes the negative supercoiling of bacterial DNA. Yamamoto and Harayama (1995. Appl. Environ. Microbiol. 61: 1104-1109) found that the frequency of base substitutions in gyrB was higher than that of 16S rRNA within the species Pseudomonas putida, thus gyrB has a higher ability than 16S rRNA to distinguish bacterial species within a genus. Species identification and detection methods based on gyrB have been developed for Bacillus spp. and Vibrio spp. (Venkateswaren et al. 1998. Appl. Environ. Microbiol. 64: 681-687; Yamada et al. 1999. Appl. Environ. Microbiol. 65: 1483-1490). There exists a need for specific primers and methods capable of specifically identifying and differentiating pathogenic Campylobacter species.